Effect of Salt Stress and Foliar Application of Salicylic Acid on Morphological, Biochemical, Anatomical, and Productivity Characteristics of Cowpea (Vigna unguiculata L.) Plants

The present study aimed to investigate the impact of salinity on vegetative growth, chemical constituents, and yields of cowpeas (Vigna unguiculata) and the possible benefits of salicylic acid (SA) on these plants after damage from salinity. To achieve these objectives, two pot experiments were carried out at the Faculty of Agriculture, Al-Azhar University, Egypt, during the two growing seasons of 2019 and 2020. The results revealed that salinity significantly decreased, and SA treatment substantially increased the plant height, number of compound leaves, number of internodes per plant, fresh weights of leaves and stems, productivity, photosynthetic pigments content, and concentrations of nitrogen (N), phosphorus (P), and potassium (K) of the cowpea plants compared with the control. The anatomical structure of stems and leaves of the plants were also investigated, and it was found that positive variations in the anatomical structure of the median portion of the main stems and blades of mature foliage leaves were detected in the stressed and SA-treated plants. In conclusion, SA treatment increased the salt stress tolerance of cowpea plants by improving the morphological and physiological attributes of the plants.


Introduction
The cowpea (Vigna ungiculata L.) is legume crop grown for its green pods or dry seeds, which provide protein, vitamins, and minerals. It is also used for other purposes such as fodder and as a green manure crop [1]. It is an important source of food for both humans and animal. Its dry seeds provide an equiponderant source of nutrients with numerous health benefits; they have high levels of protein (20.0-39.4%) and a low content of fat (3.1-30.4%) [2]. It plays a paramount role in the human diet, and nutritionists deem the supplementation of legumes through grain-based diets to be one of the best possible options for mitigating problems associated with protein malnutrition. Its area of cultivation is approximately 14 million hectares, producing 4.5 million tons of cowpeas annually in Africa, Asia, and America [3]. The cowpea has the capability of fixing atmospheric nitrogen 2.6. Determination of N, P, and K Content Samples from dried leaves were taken after 75 days from sowing of the second growing season to estimate N, P, and K as described by A.O.A.C. [36].

Anatomical Studies
Tested material included main stem and lamina of the terminal leaflet from the developed compound leaf on the median portion of the main stem were taken throughout the second growing season of 2020 at the age of 75 days from the sowing date.
Anatomical characters in cowpea stem: a.
Anatomical characters in cowpea leaf: a.
(Each value means 5 sections, 5 readings per each). The execution of microtechnique was carried out according to the method described by Nassar and El-Sahhar [37].

Statistical Analysis
We used one-way ANOVA to compare the average of the studied traits. The Duncan multiple range test as post-hoc analysis was used to show the differences among the treatments. The statistical package "SPSS for Windows ver. 25" was used to analyze the data.

Vegetative Growth Characters
Figures 1 and 2 show that the plant height and fresh weight of shoots were significantly decreased with an increase in salinity levels, and that the number of compound leaves per plant and number of internodes per plant were non-significant compared with control plants.
It can be observed in Figures 2 and 3 that the interaction between salinity stress levels and the exogenous application of SA at 100 ppm significantly increased some studied growth characters, such as plant height, number of internodes per plant, number of compound leaves per plant, and fresh weight of shoots. It is clear from Figure 3B,C that the application of SA at 100 ppm had the ability to overcome the deleterious impact of salt stress on cowpea plants, particularly those grown under 6000 ppm of salinity stress. Figure 4 shows that the salinity significantly decreased the concentration of photosynthetic pigments except for Ch a and Ch b at two low concentrations (2000 and 4000 ppm), where the rate of reduction depended on the concentration. Foliar SA applications at 100 ppm attenuated the harmful effect of salinity on photosynthetic pigments.      Figure 4 shows that the salinity significantly decreased the concentration of photosynthetic pigments except for Cha and Chb at two low concentrations (2000 and 4000 ppm), where the rate of reduction depended on the concentration. Foliar SA applications at 100 ppm attenuated the harmful effect of salinity on photosynthetic pigments.

Proline Content
There was a significantly increased amount of Pro content in the leaves of the cowpea plants with increased salinity levels, and the rate of increase depended on the concentration of salinity up to 8000 ppm ( Figure 4D). Foliar application of SA at 100 ppm decreased the free Pro concentration in the salinized plants.   Figure 4 shows that the salinity significantly decreased the concentration of synthetic pigments except for Cha and Chb at two low concentrations (2000 and 4000 where the rate of reduction depended on the concentration. Foliar SA applications ppm attenuated the harmful effect of salinity on photosynthetic pigments.

Proline Content
There was a significantly increased amount of Pro content in the leaves of the c plants with increased salinity levels, and the rate of increase depended on the con tion of salinity up to 8000 ppm ( Figure 4D). Foliar application of SA at 100 ppm dec the free Pro concentration in the salinized plants.

Proline Content
There was a significantly increased amount of Pro content in the leaves of the cowpea plants with increased salinity levels, and the rate of increase depended on the concentration of salinity up to 8000 ppm ( Figure 4D). Foliar application of SA at 100 ppm decreased the free Pro concentration in the salinized plants.

N, P, and K Concentrations
It is clear from Figure 5 that salinity at 2000 and 4000 ppm increased the concentrations of N, P, and K in the dried leaves of cowpea plants. Salinity concentrations at 6000 and 8000 decreased these concentrations significantly. The highest levels of N, P, and K were recorded in plants irrigated with 2000 ppm of salinity and sprayed with 100 ppm of SA; they had percentages of 33.11%, 45.7%, and 42.5%, respectively, more than the control plants.
are means of five replicates (n = 5), and for each parameter, the mean values ± SD followed different letter are significantly different according to Tukey's HSD range test at p ≤ 0.05 3.2.3. N, P, and K Concentrations It is clear from Figure 5 that salinity at 2000 and 4000 ppm increased the conce tions of N, P, and K in the dried leaves of cowpea plants. Salinity concentrations at and 8000 decreased these concentrations significantly. The highest levels of N, P, a were recorded in plants irrigated with 2000 ppm of salinity and sprayed with 100 pp SA; they had percentages of 33.11%, 45.7%, and 42.5%, respectively, more than the co plants.

Yield and Its Components
Results from Figure 6 show that the number of seeds per pod, number of pod plant, weight of pods per plant, and weight of 100 seeds were significantly decreas cowpeas that experienced an increase in salinity levels. The highest values of the components appeared in the control plants, whereas the lowest values were recorded salinity level of 8000 ppm. The interaction between salinity stress (at 6000 ppm) an exogenous application of SA at 100 ppm, led to a significantly increased number of s per pod, number of pods per plant, and weight of pods per plant; the percentages rea 33.5%, 25.6%, and 26.3%, respectively, over those treated with 6000 ppm of salinized ter.

Yield and Its Components
Results from Figure 6 show that the number of seeds per pod, number of pods per plant, weight of pods per plant, and weight of 100 seeds were significantly decreased in cowpeas that experienced an increase in salinity levels. The highest values of the yield components appeared in the control plants, whereas the lowest values were recorded at a salinity level of 8000 ppm. The interaction between salinity stress (at 6000 ppm) and the exogenous application of SA at 100 ppm, led to a significantly increased number of seeds per pod, number of pods per plant, and weight of pods per plant; the percentages reached 33.5%, 25.6%, and 26.3%, respectively, over those treated with 6000 ppm of salinized water.

Leaf Anatomy
Data in Table 2 and photomicrographs in Figure 8 indicate that salinity at the level of 6000 ppm decreased the thickness of the midvein, midvein bundle length and width, number of xylem rows per midvein bundle, and vessel diameter by 28.0%, 17.7%, 16.6%, 28.8%, and 14.7%, respectively, when compared with the controls.       Moreover, salinity stress at the level of 6000 ppm increased the thickness of the lamina by 66.8 % over the control. It is worth noting that the foliar application of 100 ppm of SA on cowpea plants grown under a salinity stress of 6000 ppm enhanced the anatomical features of leaves owing to an increase in their thickness in salinized plants. This treatment offset the decreases that occurred in all the included tissues of the leaf blades so that their mean values nearly reached those of the controls. It could be declared that SA had the capability of averting the deleterious impact of salinity stress on the anatomical structure of the cowpea plant leaf. That treatment decreased the effect of salt stress on the thickness of the midvein by 21.6% compared with the controls. The lamina thickness, palisade tissue thickness, and spongy tissue thickness were increased by 43.6%, 28.0%, and 18.9%, respectively, over the controls. The midvein bundles (length and width), as well as the number of xylem rows per midvein bundle and vessel diameters, were decreased by 11.0%, 10.53%, 18.6%, and 8.6%, respectively, compared with the controls. It is noteworthy that the values of all tissues in leaves of plants exposure to salinity that were sprayed with SA were definitely higher than the values for salinized plants. The thickness of the midvein, length, and width of the midvein bundle, number of xylem rows per midvein bundle, and vessel diameter were increased by 8.98%, 7.3%, 7.3%, 14.2%, and 7.0%, respectively, over the controls. The lamina was increased by 13.2% and 24.1% compared with plants treated with 6000 ppm of salinized water.

Discussion
Salt stress is considered one of the major problems in the agricultural world. It has caused severe reductions in the yield and quality of stressed plants. SA is a plant hormone that regulates plant development and diverse physiological and biochemical mechanisms in plants, especially under stress conditions. Our results show that SA mitigates the negative effects of salt stress on the growth, physiological and anatomical characteristics, and productivity of cowpea plants.
The decrease of plant growth under salt stress may be due to the reduction in cell division and elongation [38]; there is also a destructive impact of NaCl on different physiological pathways and molecular changes through impaired photosynthesis, nutrient imbalances, stomatal impedance to water flow, alterations in the ultrastructure of chloroplasts and mitochondria (which affect the natural metabolism), and hormonal imbalances [39,40]. Earlier studies [25,26,28,41] showed that SA could reduce salt damage in various plants. The exogenous application of SA is expected to control stomatal opening under stress, and this decreases transpiration and water loss, sustains turgor, and controls plant growth and productivity under stress conditions [33,42].
High levels of salinity decreased photosynthetic pigments and this pernicious influence of salinity may be due to increments of the chlorophyllase enzyme, deterioration of the photosynthetic rate, and CO 2 assimilation and stomatal motion [43]. In addition, there is a decline in chlorophyll biosynthesis intermediation concentrations [44]. During salinity stress, the increased production of oxygen radicals in cells causes oxidation and, hence, the degradation of photosynthetic pigments [45]. SA reduces the harmful effect of salinity on chlorophyll by decreasing the ROS and elevating the antioxidant systems. This enhances plant growth by activating cell division and elongation and perhaps by inhibiting chlorophyll oxidase enzymes, subsequently preventing chlorophyll breakdown and enhancing photosynthesis [46,47].
Salinity stress increases the Pro content and this increases an important mechanism to reduce the harmful effect of salt stress and improve plant growth [48]. Earlier reports [49][50][51] cleared the accumulation of Pro under salinity. The foliar application of SA under salinity may reduce the devastating effect of salt stress on plant growth and ameliorate Pro accumulation [52].
In this research, the results show that increased salinity of up to 8000 ppm decreased concentrations of N, P, and K ( Figure 5). This may be due to an ionic imbalance [26] resulting from a disorder in the integrity of the plasma membranes of cells [53] in addition to a converse relationship between Na and other elements [54]. SA influences numerous important physiological functions in plants, such as increasing the uptake of nutrients and decreasing Na + concentrations under salt stress [22,23]. Similarly, [26] researchers have reported that increasing the concentration of SA up to 90 ppm mitigates the negative effect of salinity on N, P, and K concentrations and decreases the Na + content of strawberry leaves under high levels of NaCl (40 mM). It was reported that the foliar supplementation of SA at 0, 1.0, 1.5, and 2.0 mM reduced the Na uptake of plants and was efficient in enhancing nutrient uptake by cotton seedlings grown under 150 mM NaCl [28]. Moreover, SA at 1.0 mmol/L plus NaCl treatment significantly improved nutrient accumulation and also decreased Na + and Cl − accumulations compared with NaCl treatment [29].
A decrease in yield under salinity conditions was probably due to the reduction in photoassimilation assembly, as well as photoassimilation mobilization, which induced a decrease in the harvest index [55]. The reductions of yield may be due to the reverse impact of salt stress on growth characteristics and physiological operations such as water uptake, photosynthesis, flowering, and pod formation, which led to a decreased yield [52]. Nevertheless, in the present study, SA application ameliorated salinity tolerance in cowpeas and enhanced the plants' yields. The function of SA in yields is probably due to its fundamental role in stress tolerance. It decreases oxidative damage and increases plant growth and yields under salinity stress [56], and it also improves nutrient uptake and increases the levels of chlorophyll and carotenoid pigments, modulates the activities of several enzymes, and conserves the integrity of cell membranes [22,23]. It also plays a role in balancing plant hormones [57].
Anatomical characters were decreased by salinity in cowpea plants. Research by [58] found that salinity decreased the thickness of the upper epidermal layer, cortex layers, hypodermal layer, and pith area. It was noticed that spraying 100 ppm of SA on cowpea plants exposed to 6000 ppm of salinity stress enhanced anatomical features of the main stem. A study by [59] found that drought stress at 40% or 20% reduced the upper and lower epidermis, midvein thickness of the leaflet blade (in length and width), number of xylem rows per midvein bundle, and vessel diameter of cowpea plants. Salinity at 6000 ppm decreased the thickness of both the lamina and midvein of the mature foliage leaf of the basil plant [18].
SA had the capability of avoiding the deleterious impact of salt stress on the anatomical structure of the cowpea plant. In the present study, the foliar application of 100 ppm of SA on cowpea plants exposed to 6000 ppm of salinity stress enhanced the anatomical features of leaves. These results are in accordance with those observed by [30].

Conclusions
The results of this study revealed that the exogenous spraying of SA might alleviate the inhibitory impact of salinity stress on the growth, physiological and anatomical features, and the productivity of cowpea plants.